Ultrasound device

ABSTRACT

The invention relates to an ultrasound device comprising a piezo element ( 4 ), which generates ultrasound waves, and intermediate elements ( 5, 6 ) via which the ultrasound waves are transmitted to the probes ( 7 ) and into a sample volume inside a microplate ( 1 ). A piezo element ( 4 ) comprises a number of probes ( 7 ), which radiate the ultrasound and which are arranged next to one another in a row. Between the point of origin of the sound wave on the piezo element ( 4 ) and the point of output of the sound wave on the radiating probes ( 7 ), the wave-transmitting elements ( 5, 16 ) do not widen at all with regard to the surface of the piezo element ( 4 ).

The invention relates to an ultrasound device, specially an ultrasounddevice for the disintegration of cells or cellular material.

In the context of biological and pharmaceutical testing tendency goes tosmall sample quantities, worked on automatically with high throughput instandard microplates, also called multi-well-plates. These microplatesexhibit sample containers, called wells, in a number of between 6 (2×3)and 9600 (80×120) with volumes of millilitres down to picolitres. Theplates possess a fixed outer size of approximately 85×128 mm with aprecise arrangement of the sample containers (wells). External size andthe arrangement of the wells usually follow the international ANSIstandard.

In order to examine biological cell material the cells have to bedisintegrated, which means that the cell walls must be opened ordestroyed, in order to get to the material located inside of the cell.This cell disintegration has to be performed as carefully as possibleand in a way, that the addition of foreign substances to the sample canbe avoided.

With ultrasonic it is possible to disintegrate cells in small volumes,below one millilitre, rapid and without the addition of foreignmaterial. The cells suspended in a liquid medium are destroyed byultrasonic waves of low frequency and high power.

High frequent acoustic waves with high amplitude cause the formation ofsmall bubbles in liquids, which first increase, until they implode. Thiseffect, called cavitation, leads to the disintegration of membranes andcell walls by the arising fast changes in pressure. The cavitation isstronger in the range of low frequencies than in the range of highfrequencies, so that for the disintegration of cells ultrasonic waveswith frequencies as low as possible should be used.

Usually the applied ultrasonic frequencies are about 20 kHz, since therange is limited downwards by the threshold of audibility.

An ultrasonic device for such an application consists of a generator,which produces an electrical output wave (sinus wave) with a frequencyof for example 20 kHz, an ultrasonic electrical transducer, which isusually of the piezoelectric type, and converts the electrical outputwave from the generator into a mechanical motion perpendicular to thesurface of the ultrasonic electrical transducer, a mechanical transducer(impedance transducer), which forwards the ultrasonic energy generatedat the electrical ultrasonic transducer, as well as an ultrasonic hornand/or a probe, which focuses the ultrasonic power and directs it intothe liquid with the sample.

The oscillating probe causes extremely high acoustic pressurefluctuations in the liquid at its tip, which are responsible for thephenomenon of cavitation.

Horn and probes serve, as mentioned, for the transmission of ultrasonicinto the sample. They cause thereby, dependent on their geometries, anincrease of the intensity: The intensity of ultrasonic irradiation intothe medium increases upon decreasing the final diameter at the end pointof the tip. However, it is not possible to transmit any desired level ofamplitude simultaneously with high acoustic power into the medium.Moreover, the size of the tip has to be adapted to the size of thesample tube. For this reason the probes and tips have to decrease indiameter towards the end, if they are operated in the small volumes of amicroplate.

The geometry at the end of the tip also determines the radiationbehavior. An even surface of the tip perpendicularly to the longitudinaldirection causes a strong radiation in forward direction; a conical tipcauses a stronger lateral radiation.

From the U.S. Pat. No. 6,071,480 an ultrasonic device is known, in whichmicro vessels are arranged in the maximal amplitude of the transversalwave, which has for instance the double frequency of the longitudinalwave. In the end plate of such an ultrasonic horn are holes for themicro vessels, into which the micro vessels are inserted. Thisarrangement has the disadvantage that it cannot be used for standardizedsample container arrangements, because the amplitude maximums on thesurface of the end plate possess geometrical figures in the kind ofcircles and not in a the rectangular pattern of the microplatesmentioned. For this reason the ultrasonic device known from U.S. Pat.No. 6,071,480 can be only used in connection with the described,separate micro sample containers.

So far samples are disintegrated in microplates with ultrasonic bydipping the tip of a probe manually into each particular well. Thistechnique of disintegration is time consuming and cannot bestandardized.

The used of multi-element-probes has previously been tried. To animpedance transducer, in the form of a relatively broad block severaltips are arranged side by side in a row. To this the electrictransducer, e.g. a piezo element, is connected via a relatively narrowcoupling transducer. By this arrangement the problem arises, thatalready with few tips the distribution of ultrasonic intensity isuneven.

In U.S. Pat. No. 4,571,087 a device for the positioning of eachindividual well of a microplate above an ultrasonic horn is described.From the ultrasonic probe, located perpendicularly under the well of amicroplate, the ultrasonic power is transferred into the well via aliquid bath, usually a water bath, while the microplate is moved by thedevice in x-y-direction. This device has the disadvantage, that theindividual wells of the microplate can only be treated separately oneafter another with ultrasonic, which is time consuming and an highenergy transfer into the samples is not possible.

Purpose of the invention is it to create an ultrasonic device for theacoustic irradiation of media in microplates, or similar arrangement oftubes, or also in chips, with which an even acoustic irradiation of awhole set of containers etc. is possible.

This task is solved according to the invention with the ultrasonicdevice indicated in the patent claim 1. Favorable arrangements aredescribed in the sub-claims.

In the ultrasonic device according to the invention therefore, nowidening of the wave transmitting elements occurs between the origin ofthe sound pressure wave at the ultrasonic electric transducer, and theemitting place of the sound pressure wave at the ultrasonic emitter. Itshowed up, that a widening causes a disturbance in the wave propagation,which leads to an uneven amplitude distribution.

Finally, all elements by which the sound moves, are essentially locatedwithin the active surface of the ultrasonic electrical transducer.

The invention is particularly favorably applicable with ultrasonicdevices, with which several sound-delivering elements are arranged in arow and/or a surface next to each other.

Instead of a linear arrangement also a two-dimensional arrangement ispossible. So the arrangement can be square for example. Also transitionsto round or rectangular arrangements are possible. Important is aboveall, that within the entire arrangement no transverse forces ortransverse vibrations, i.e. no transverse-waves or no bending-vibrationsare developed. Thus the surface has to be excited into a relativelyeven, longitudinal oscillation.

The same applies not only to the sound-emitting elements, but also tothe sound-generating ultrasonic electrical transducer which can likewisebe arranged in a majority, and/or in different linear and/or intwo-dimensional ways.

With the ultrasonic device according to the invention both, a directacoustic irradiation of a microplate located below the sound-emittingelement or an indirect acoustic irradiation of a microplate, which liesabove the sound-emitting element, are possible. The plate can be cooledduring the direct acoustic irradiation.

On the market so far no ultrasonic device is available, which would besuitable for a fast and reproducible disintegration of cells inmicroplates. The ultrasonic device according to the invention solvesthis problem. It is possible thereby to achieve a rapid, reproducibledisintegration directly in the microplate, which is necessary for thestandardization and certifying of tests. The ultrasonic device accordingto the invention offers all possibilities for automation and can, incombination with other devices, be used in high throughput processes.

The ultrasonic device according to the invention for sonic irradiationof microplates can find applications within several domains of pharmacy,biotechnology, diagnostics, environmental technology, microbiology,immunology, cell biology and medicine. Examples for applications coverapart from the disintegration of biological material, e.g. tissues,cells, bacteria, cell material, organelles, aggregates, viruses,high-throughput-screening, toxicity studies for sample preparation inenzymatic tests, ELISA's, RIA's, genomics and proteomics, PCR and/orRT-PCR, DNA- or RNA-labeling, hybridizing, receptor-binding-studies foracceleration, catalysis and increase of the yield of chemical reactions,production of liposomes, micro-emulsions, nano-particles and suchlike aswell as for suspending, homogenizing, emulsifying and extracting andothers.

On the basis of the drawings the invention is described exemplarily.They show:

FIG. 1 a standardized micro plate;

FIG. 2 an ultrasonic horn for the microplate of FIG. 1 in a viewparallel to the longitudinal axis of the ultrasonic horn;

FIG. 3 the ultrasonic horn of the FIG. 2 in a view transverse to thelongitudinal axis;

FIG. 4 a view similarly to FIG. 3, whereby two ultrasonic horns arearranged in longitudinal direction next to each other; and

FIG. 5 a device for the indirect irradiation of the microplate of FIG. 1in a side view.

In FIG. 1 a microplate according to ANSI standard is shown. In thesestandardized microplates (1) with the external dimensions of 85mm×127.76 mm are the wells (2) for the samples, arranged in such amanner, that the number of wells in horizontal direction (inx-direction) is an integral multiple of three and in vertical direction(in y-direction) an integral multiple of two. The presently mostly used96-well-microplate, shown in FIG. 1 exhibits 12 wells in horizontaldirection 8 and in vertical direction. The inside diameter of the wells(2) is in each case 6 mm in a 96-well-platte.

An ultrasonic horn for the direct acoustic irradiation of a number ofwells (2) of a 96-well-mikroplatte (1) can contain 4 probes next to oneanother, for example. With two of those ultrasonic horns, which arearranged in longitudinal direction next to each other, it is possible toirradiate a complete row of wells (2) of the microplate (1) iny-direction.

FIG. 2 shows an ultrasonic probe (3) for the microplate (1) in a viewparallel to the longitudinal axis of the ultrasonic horn, i.e. thedrawing plane is perpendicularly to the longitudinal axis. FIG. 3 showsan ultrasonic horn (3) with the axis rotated by 90°. As shown in FIGS. 2and 3, the ultrasonic horn (3) is constructed as follows:

A piezo element (4) forms the core of the ultrasonic horn (3). The piezoelement (4) converts the electrical waves or impulses from a generator(not shown) into mechanical impulses (acoustic waves, ultrasonic waves).To the piezo element (4) in the irradiation direction an impedancetransducer (5) is connected, which has a length of a quarter wave. Tothe impedance transducer (5) an ultrasonic horn (6) is connected, whichin one dimension linear tapered in a conical way and causes a firstfocusing of the ultrasonic power on a rectangular area. The ultrasonichorn (6) is three-quarter of the wave long. The narrow end of theultrasonic horn (6) is connected to ultrasonic probes (7), eachpossessing at the end a quartz tip (not shown) fixed with glue.

Form and structure of the ultrasonic horn (6) and the probe (7) arearranged in such a way, that a standing wave is formed. At end face ofthe tip of the probe (7) the ultrasonic power should be emitted ashomogeneously as possible. This is ensured best by a rod with an evenend face, which is evenly brought to oscillations over its whole width,in order to avoid bending-vibrations. In addition the probes areequipped with replaceable quartz tips. During the transition to thequartz the stage reduction should be as small as possible, in order toavoid breaking of the quartz.

Usually aluminum and quartz are the preferential materials for thesound-transmitting parts of the ultrasonic head (3), however obviouslyalso other materials are general usable, as long as they possesscomparable impedance factors.

The piezo element (4) generates ultrasonic waves with a frequency oftypically 20 kHz and with energy sufficient for cavitation in the wells(2) of the microplate (1) and which is also sufficient to disintegratecells or cellular material.

An end piece (8), which is arranged behind the piezo element (4), makesa tightening of the piezo elements (4) between the end piece (8) and theultrasonic horn (6) possible by means of a screw (9). The screw runsthrough the end piece (8), the piezo element (4) and the impedancetransducer (5) and is screwed into the ultrasonic horn (6).

The end piece (8), the piezo element (4), and the impedance transducer(5) are cylindrical and possess all the same diameters. This diameter is35 mm, for example, for the ultrasonic head (3) used in a standard96-well-microplates (1).

Alternatively, the end piece (8), the piezo element (4), and theimpedance transducer (5) can possess also other forms, e.g. they can besquare or rectangular in its cross section.

To form the ultrasonic horn (6) either a round or a square column can beused. The side of the square has to be similar to the diameter of theend piece (8), the piezo element (4) and the impedance transducer (5).Alternatively, a cylinder with the same diameter as these parts, e.g. 35mm, can be used.

In the dimension transverse to the longitudinal direction the ultrasonichorn (6) tapers itself, as shown in FIG. 2, from the full edge lengthand/or the full diameter to a width, which is about the width,respectvely the diameter of a probe (7) or it is slightly larger.

In the example described, the ultrasonic horn (6) tapers itself to anarea of 35 mm×9 mm.

FIG. 3 shows a front view on the ultrasonic horn (3). It is shown thatin the longitudinal direction along the centre line of the ultrasonichorn (6), four probes (7) are inserted into the ultrasonic horn (6). Thedistance of the tips of the probes (7) corresponds exactly to thedistance of the wells (2) in the microplate (1).

Impedance transducer (5), the ultrasonic horn (6) and the part of theprobe (7), into which the quartz tip is inserted, consist preferably ofaluminum or an aluminum alloy, which exhibits good sound transmissioncharacteristics. The end piece (8) consists preferably of brass andalternatively of steel or tantalum.

The quartz tips of the probes (7) can possess a diameter of 2 mm for theuse in microplates with up to 384 wells. Using microplates with a higheramount of wells the diameter has to be reduced according to the size ofthe wells. The form of the tip can be linear as a rod or conicallytapering, particularly for higher energy entries.

As shown in FIG. 4, two of such ultrasonic heads (3) can be arranged inlongitudinal direction next to each other, whereby the arrangement takesplace in a manner that the distance between all probes (7) is the sameand corresponds to the distance of the wells (2) in the microplate (1).With such an arrangement a complete row of wells (2) can be treated atthe same time.

Alternatively, also a common ultrasonic horn (6) can be used for twopairs of piezo elements, two end pieces, two impedance transducers(exciter arrangements) (4, 5, 8) and eight probes (7), in the exampledescribed. In this case the ultrasonic horn (6) consists of a plate withoblong-rectangular basic form, and their length is essentially equal tothe overall length of the exciter arrangements next to one another (4,5, 8). The thickness of the horn is equal to the exciter arrangements(4, 5, 8) and is tapering towards the probes (7) according to theillustration in FIG. 2. With such an ultrasonic head the exciterarrangements (4, 5, 8), and the probes (7) are in each case arrangedalong the centre line of the elongated ultrasonic horn (6).

Such ultrasonic heads can also be arranged next to each other in such away that a two-dimensional array of probes is formed, with which a wholemicroplate can be treated at one time. Alternatively, it is possible,for example, to treat each second row of wells (2) in the microplate(1). Naturally also arrays for the treatment of half etc. microplate canbe manufactured.

The number of exciter arrangements (4, 5, 8) and the number of probes(7) at a common ultrasonic horn (6) is arbitrary in each case and can beselected with consideration of the intended application. Equally, asmany ultrasonic horns (6) as desired can be arranged next to each otheror can be interconnected, in order to form linear and/or two-dimensionalarrays.

Also with two-dimensional array arrangements a common ultrasonic horn(6) can be planned, with all exciter arrangements (4, 5, 8), and probes(7), whereby the exciter arrangements (4, 5, 8) and the probe (7) form arectangular arrangement in each case.

The focusing of the ultrasonic power within the range of the ultrasonichorn (6) can be achieved by different geometrical arrangements of thehorn (6). Possible is once a stacked form, by which the cross section ofthe horn (6) decreases by steps. Moreover, an exponential form ispossible, in which the cross section of the horn (6) decreasescontinuously in an exponentially way. Finally a conical form ispossible, in which the cross section over the length decreases in alinear way. This type is very stable and simple to manufacture and istherefore preferred, although the focusing effect is smaller than withthe other two arrangements.

Essential is in all arrangements, that between the origin of theacoustic wave at the respective piezo element (4) up to the tip of theassociated probe (7) essentially no widening of the wave-transferringparts arises, even if it is reduced again. In no position,perpendicularly to the sound propagation, the sound-transferring partsshould possess a cross-section area, which is substantially larger thanthe surface of the piezo elements (4) and/or the ultrasonic transducer.If necessary, a widening of 20 to 30% is permissible at the transitionto the piezo element (4). Possible are also small recessing in the soundtransmitting parts for example for the attachment of fixtures. Of courseone has to take care that the fixing points are always at the nodes ofthe wave and not at the antinodes.

It is also important, that the tips of the probes are centricallyarranged, that means in the case of a linear arrangement on the centreline of the ultrasonic horn (6), of the impedance transducer (5) and thepiezo elements (4).

The arrangement described can be supplemented by mechanisms forautomatic moving and shifting of the ultrasonic head (3) and/or themicroplate (1) in the three directions in space.

Alternatively to the arrangement described for the direct ultrasonicradiation of the samples in microplates by immersing the tips into thesample liquid from above also an arrangement for indirect radiationthrough the bottom of the microplate can be planned. A part of theultrasonic power is absorbed by the bottom of the microplate. Howeverthe movement of the ultrasonic tips towards the sample and the cleaningof the ultrasonic tips after each treatment are not necessary in thisarrangement.

An even indirect irradiation of the entire microplate is only possibleby excitatation with a number of piezo elements vibrating in phase. Inthe ideal case this is performed by a number of independently vibratingtransducers whose number corresponds to the number of wells exposed tosound. The limit, up to which this is possible in practice, is reachedwith a 96-well-plate.

A more general construction, which can be used for all microplates,consists of a metal plate on which the microplate is put. The metalplate is brought to evenly vibration over the whole area by a number ofpiezo elements located below the plate, covering its whole lower area.

The structure of such an arrangement, representing a second form of theultrasonic device, is shown in FIG. 5. In principle this structurecorresponds to an arrangement according to FIGS. 2 and 3 turned upsidedown, with a distribution of the piezo element (4) over the whole area.The probe (7) is replaced by a metal plate (10) and the ultrasonic hornis replaced by a transmission cylinder (16).

In detail, the arrangement shown in FIG. 5 consists of the end piece(8), the piezo element (4), and the impedance transducer (5). The endpiece (8), the piezo element (4) and the impedance transducer (5) arescrewed onto the solid transmission cylinder (16). At the other end ofthe transmission cylinder (16) the metal plate (10) is fastened. Themetal plate (10) is covered with a number of excitation and transmissionarrangements (4, 5, 8, 16), in such a way, that only little gaps remainbetween each individual excitation and transmission arrangement (4, 5,8, 16). In other words, the metal plate (10) is closely occupies withexcitation - and transmission arrangements (4, 5, 8, 16).

The diameter of the transmission cylinder (16) corresponds in each caseto the diameter of the piezo element (4). However, it not tapers itself,as this is the case with the ultrasonic horn (6) of the first type.Again is important, that no broadenings occur in the direction of theacoustic waves between the piezo element (4), the beginning of thetransmission cylinder (16) and the metal plate (10). It is guaranteed,that no substantially broadening of the sound transmission occur, alsoat the transition of intermediate cylinder (16) to the metal plate (10),by close covering the metal plate (10) with the excitation andintermediate elements (4, 5, 8, 16).

The whole arrangement is so dimensioned, that the end plate, from whichthe ultrasonic wave is emitted into a liquid or to the bottom of themicroplate, is located at the amplitude maximum, i.e. at an integralmultiples of the lambda/2 wave. Attachment- and transition points shouldlie in the nodes of the sonic wave.

The piezo elements (4) must vibrate with the same energy in phase, inorder to irradiate all samples in the wells (2) of the microplate (1)with the same ultrasonic power.

The microplate can be directly put on the surface of the metal plate(10) or into a bath inside the metal plate (10). The external dimensionsof the metal plate (10) correspond to the external dimensions of themicroplate (1) plus an edge. A liquid bath is necessary to radiateultrasonic energy into wells in U- or V-form. If the bottom of themicroplate is planar it can be put on the metal plate (10) withoutadding a liquid. Without liquid the sound transmission can be improvedby a Mylar foil (Mylar is a registered trade mark of the DuPont groupfor a polyester foil) or a liquid film with high viscosity.

To avoid splashes, the microplate can be covered with a foil. During thedirect acoustic irradiation this foil can be simply punctured by thetips. Thus each wells of the microplate is covered, and the neighboringwells cannot be contaminated during the treatment with ultrasound.

Preferably, the ultrasonic power irradiated into the sample volume ismeasured and the measurement result is used for the regulation of theenergy emission. Thus it is also possible to irradiate with a slightlyhigher energy at the beginning of the treatment with ultrasound anddown-regulate afterwards in such a way, that the energy level emittedinto the sample volume remains constant. Favorably a sensor, p. e. afurther ultrasonic electric transducer is attached at the sample, forinstance in form of a piezo element, which measures the acousticpressure irradiated into the sample volume as an electrical signal. By asensor attached directly to the sample and/or the microplate it ispossible to measure the amplitude of the irradiated ultrasonic wavedirectly at the sample and keep it constant by an appropriateregulation.

Measurement and regulation of the irradiated amplitude or energy is alsopossible by measuring the pressure, the force, or simply by an increaseof weight at the sample/s volume. In case of a direct radiation themicroplate can be simply put on a balance.

1. Ultrasonic device marked by an ultrasonic electrical transducer (4),which generates ultrasonic waves, which are transferred via intermediateelements (6, 16) into a sample volume (2), wherein an even longitudinaloscillation develops over several ultrasonic radiating elements (7) or asurface (10) and thus causing an even irradiation of ultrasonic intoseveral sample containers simultaneously, caused by an arrangement, thatfrom the generating place of the acoustic wave at the ultrasonicelectrical transducer (4) via the wave-transmitting intermediateelements (6, 16) up the radiating element (7, 10) emitting the acousticwave to the sample, at essentially no widening arises in relation to thegenerating place of the acoustic wave.
 2. Ultrasonic transducer definedin claim 1, wherein for each ultrasonic transducer (4) severalultrasonic radiating elements (7) are available.
 3. Ultrasonic devicedefined in claim 1, wherein for one ultrasonic radiating elements (10)several ultrasonic electric transducers (4) without or with intermediateelements (5, 16) are available.
 4. Ultrasonic device defined in claim 2or 3, wherein for several ultrasonic electrical transducers (4)respectively several ultrasonic radiating elements (7, 10) a commonintermediate element (6) is available.
 5. Ultrasonic device defined inclaim 1, wherein the ultrasonic transducer is a piezo element (4). 6.Ultrasonic device defined in claim 2, wherein the ultrasonic emittingelements are probes (7) immersing into a sample liquid.
 7. Ultrasonicdevice defined in claim 2, wherein the wave-transferring intermediateelements (5, 6) are preferentially made of aluminum or aluminum alloysand the radiating elements (7) preferentially made of aluminum oraluminum alloys with quartz tips tips.
 8. Ultrasonic device defined inclaim 3, wherein the ultrasonic radiating element is a metal plate (10).9. Ultrasonic device defined in claim 1, wherein the sample volume (2)is contained in a sample container in a microplate (1), or in containersof similar arrangement, which can correspond also to parts of amicroplate.
 10. Use of an ultrasonic device according to claim 1 to 9for disintegration of biological material for the sample preparation ofPCR, genomics and proteomics, for sample preparation in enzymatic tests,hybridization- and receptor binding studies, for the acceleration,catalysis and increase of the yield of chemical reactions, forgenerating liposoms, micro emulsions or nanopaticles, as well as forsuspending, homogenizing, emulsifying and extracting.